ABSTRACT: The brain simultaneously processes tremendous amount of information using billions of neurons. Technologies for large scale recording and modulation of neurons in the brain of behaving animals sought by this RFA should provide researchers with tools to correlate neural circuits with behavior. To identify and control the circuits responsible for a specific state of behavior, technologies allowing activity-dependent labeling of neurons during a restricted time-window are required. Current methods do not allow direct labeling of activated neurons at a large scale with fast temporal resolution. Techniques for labeling neurons expressing immediate early genes at a time of drug exposure or withdrawal are indirect and slow (hours), because they depend on drug delivery and washout. Approaches that couple light illumination and calcium signaling to photoswitching of a fluorescent protein or gene expression are faster and direct, but use potentially toxic violet or blue light. This light poorly penetrates tissue, requires invasive procedures for delivery to the brain, and illuminates only small volume (0.5 mm3). Here we propose to develop a novel technology that couples calcium signaling, directly associated with neuronal activity, with illumination by near-infrared (NIR) light, which defines the time window for labeling. Because NIR light (740-780 nm) was shown to penetrate deep into the mouse brain, the proposed technology should allow timely precise, direct recording of active neurons in the whole brain of freely behaving mice. As a NIR light-sensing module, we will use a NIR optogenetic system of light-inducible protein-protein interaction (PPI) recently developed in the lab and validated in neurons. The proposed system will sense a coincidence of NIR light-induced PPI and calcium-induced PPI to produce expression of any transgene. This will allow fluorescent labeling of activated neurons for their visualization and production of optogenetic tools for their further manipulation. We will use our expertise in engineering of NIR light-sensing tools to develop the proposed system and systematically optimize it in cultured cells and primary neurons. We then will extensively characterize it, establish its cross-talk free combination with opsin-based actuators and, moreover, validate it in vivo. The proposed technology should reveal functional neural circuits across different brain structures that underlie specific stages of behavior or cognitive process, such as exploring a new object or learning a new task. PUBLIC RELEVANCE: Non-invasive technologies for timely precise whole-brain recording and control of functional neural circuits in freely behaving animals should facilitate our understanding of how brain processes information in health and disease, and eventually lead to development of new therapies for brain disorders.